Heterologous Expression of SFA Genes

We used N-terminal FLAG pPXV plasmid (modified plasmid, courtesy of Dr. W. John Haynes, University of Wisconsin, Madison, WI, USA) to clone 13 different SFA genes (SFA1a, SFA2, SFA3, SFA4, SFA5a, SFA6b, SFA7a, SFA8a, SFA9, SFA10a, SFA11a, SFA12a and SFA13a). All the gene sequences are available in ParameciumDB (http://paramecium.cgm.cnrs-gif.fr/). Accession numbers are available in Additional file 2: Table S1. All the primers used for FLAG-epitope tagging are listed in Additional file 2: Table S2. The target gene sequence was amplified by using Q5TM Hot Start high fidelity DNA polymerase (New England BioLab Inc, Ipswich, MA, USA) according to the manufacturer’s protocol. Resulting amplicons were inserted into the pPXV plasmid using restriction enzymes (NheI/KpnI or ApaI/SacI) (New England BioLab Inc, Ipswich, MA, USA) and the amplicon sequences were confirmed by sequencing. All the FLAG pPXV plasmids containing the target gene sequences were linearized by using NotI restriction enzyme (New England BioLab Inc, Ipswich, MA, USA) and injected as previously described [30 (link)]. The presence of pPXV-3XFLAG-SFA in individual clones was confirmed using PCR with extracted genomic DNA as a template.